EPR spectroscopy and spin-trapping methods were used to identify and monitor the formation and utilization of free radicals. We studied the Cu,Zn-superoxide dismutase mutants found to be associated with familial amyotrophic lateral sclerosis. We have shown that, in addition to the known dismutase activity, Cu,Zn-SOD also catalyzes the generation of free radicals with hydrogen peroxide as substrate. In the current study with the wild-type and two mutants, G93A and A4V, we found that all three enzymes possess identical dismutase activity; however, free radical- generating activity is enhanced with the mutant enzymes when hydrogen peroxide concentration is relatively low. This is due to a decrease in the value of Km for hydrogen peroxide in the order of wild-type>G93A>A4V, while k(cat) remains the same for all three enzymes. Our finding that A4V has a lower Km value is in accord with the clinical observation that A4V patients have the most aggressive form of FALS. Nitric oxide reacts with superoxide anion to form peroxynitrite under oxidative stress. We showed that peroxynitrite-mediated tyrosine nitration, which is enhanced by Fe(III)EDTA or Cu,Zn-SOD, occurred on the tyrosine residues of cdc2(6-20)NH2, which is a good peptide substrate for lck kinase, a src-tyrosine kinase, and the nitrated tyrosine cannot be phosphorylated. To test the effect on the regulatory function due to tyrosine nitration, we studied the binding capacity of two src homology 2 (SH2) recognition peptides to the SH2 domains of phospholipase C type gamma (PLC-gamma). We found that the tyrosine-phosphorylated peptide binds to PLC-gamma with high affinity, while the nitrated peptide fails to bind PLC-gamma. Thus, the irreversible tyrosine nitration will permanently impair the regulatory function of cyclic cascades involving tyrosine phosphorylation. Mechanistic study of c-jun ubiquitination catalyzed by E2-20K or by E2- 14K/E3 indicates that with the E2-20 system ubiquitinylation proceeds with a normal stepwise dissociative mechanism, while the E2-14K/E3 system favors an associative processive mechanism. Mechanistic study on the electric field-induced transient membrane pore formation indicates that a significant reduction in either the number or size of pores occurs within the first few hundred msec after the pulse. With this method, we showed that hydrogen peroxide is generated during EGF-induced signal transduction and it functions as a second messenger to mediate the extent of tyrosine phosphorylation on various cellular proteins and intracellular Ca mobilization.